FVIII-specific suppressive capability of CD4⁺CD25⁺ T cells derived from FVIII plasmid + anti-CD3–treated mice. For the FVIII-specific suppressive assay, we used as responder CD4⁺ T (Tresp) cells from the spleen of a FVIII plasmid only–treated mouse at week 4 after plasmid injection. CD4⁺CD25⁺ T cells from pooled splenic cells of naive hemophilia A mice or FVIII plasmid + anti-CD3–treated mice were used as suppressive cells. The final coculture system consisted of 0.8 × 10⁵ CD4⁺ Tresp cells, 1.5 × 10⁵ irradiated CD4⁻ cells, and CD4⁺CD25⁺ Treg cells at the indicated Treg/Tresp ratio. (A) CD4⁺CD25⁺ Tregs were isolated at week 2 after plasmid injection from mice treated with FVIII plasmid + anti-CD3. (B) CD4⁺CD25⁺ Tregs were isolated at week 6 after plasmid injection from mice treated with FVIII plasmid + anti-CD3. Data shown are mean ± SD of counts per minute of [³H]thymidine incorporation in triplicate wells.